Validation of the Bioequivalence of USP Potency Adjusted Porcine, Ovine, and Bovine Heparins

Introduction: Currently, there is a shortage of porcine heparin due to several factors such as limited availability of porcine mucosa, supply chain issues, and increased usage due to COVID-19. This has warranted the development of heparin from alternate sources such as bovine and ovine mucosa which is abundantly available for this purpose. On a mass basis, commercially available porcine heparins exhibit a similar potency (200 units/mg) to their ovine counterpart (190 units/mg) and a higher potency in contrast to their bovine counterpart (130-150 units/mg). Therefore, at gravimetric levels, the porcine heparins exhibit stronger biochemical and pharmacological effects in various laboratory assays in comparison to bovine heparin and similar effects in comparison to ovine heparins. Since heparin is standardized in biologic units and cross referenced against USP or EP Standard, it is hypothesized that potency equated porcine, ovine, and bovine heparin will exhibit similar biologic activities in laboratory assays carried out in the in vitro setting. The purpose of this study is to compare the biologic properties of the porcine, ovine, and bovine heparin at USP potency equated levels in standardized laboratory assays. Materials and Methods: Active pharmaceutical ingredients (API) of porcine mucosal heparin (200 units/mg) of U.S. origin was commercially obtained from Medefil Inc. (Glendale Heights, IL). Ovine heparin was obtained from Ronnsi Pharmaceutical (Jiangsu, China). Bovine heparin (140 units/mg) was obtained from Kin Master Pharmaceuticals (Posso Fundo, Brazil). All heparins were diluted at a concentration of 100 units/mL in saline. The anticoagulant effect of all heparins were evaluated using the whole blood clotting assays such as the ACT and thromboelastographic methods. Heparins were diluted in citrated human plasma yielding a final concentration range of 0-1 unit/mL. Clot based assays such as aPTT, TT, and prothrombinase induced clotting time (PiCT) were measured. Thrombin generation inhibition assay was carried out using a kinetic assay (CAT system, Diagnositca Stago, Paris, France). Protamine and heparinase neutralization profiles of these agents were also investigated in the plasma-based systems. These assays were then repeated at gravimetric dosages at final concentrations of 0-10 ug/mL. The results collected from these trials were then mathematically converted to units and compared to the results collected from the potency adjusted trials. All results were tabulated and compared, and applicable statistical methods were applied. Results: The USP potency adjusted heparin exhibited comparable anticoagulant effects in both the ACT and TEG assays. At equigravimetric levels porcine and ovine heparins produced comparable anticoagulant effects and bovine heparin produced weaker anticoagulant effect in both assays. In the citrated plasma supplementation studies, all drugs produced similar anticoagulant effects at potency adjusted dosages. In the chromogenic anti-Xa and anti-IIa assays, the behaviors of the agents were also comparable. In the thrombin generation assays, in terms of peak thrombin generation, area under the curve, and lag time, the porcine, ovine, and bovine heparins showed comparable effects. The protamine neutralization profiles of the porcine, ovine, and bovine heparin exhibited variable assay dependent results. Potency adjusted bovine heparin required higher amount of protamine for the complete neutralization of the biologic effects in comparison to the porcine heparin. At gravimetric concentrations, bovine heparins exhibited lower potencies than both the porcine and ovine heparins, which produced similar results. Summary and Conclusion: These results show that at potency adjusted concentrations, the porcine, ovine, and bovine heparin exhibit comparable biochemical and anticoagulant responses in the plasma-based systems. Therefore, the hypothesis that potency equated porcine, ovine, and bovine heparins exhibit comparable biochemical and anticoagulant activities is validated. Thus, the proposed approach to standardize heparins against a common standard in a biologic assay such as the USP method is valid. Furthermore, these results warrant regulatory considerations to fast track the review process for the re-introduction of bovine heparin and approval of bovine heparin as a biosimilar anticoagulant to porcine heparin. Disclosures No relevant conflicts of interest to declare.

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Generic and Branded Versions of Argatroban Exhibit Differential Anticoagulant Effects In Whole Blood, Plasma Based Assays and Thrombin Generation Assays

Blood ◽

10.1182/blood.v116.21.5129.5129 ◽

2010 ◽

Vol 116 (21) ◽

pp. 5129-5129

Author(s):

Jawed Fareed ◽

Debra Hoppensteadt ◽

Omer Iqbal ◽

Jeanine M. Walenga ◽

Bruce E Lewis

Keyword(s):

Protein Interactions ◽

Whole Blood ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Anticoagulant Effect ◽

Thrombin Time ◽

Clotting Time ◽

Generic Products

Abstract Abstract 5129 Several generic versions of argatroban) (Mitsubishi; Tokyo, Japan) have been introduced in Japan (Argaron, Gartban, Slovastan). In addition, other generic versions of argatroban are being considered by the European and North American regulatory bodies. While the generic versions of argatroban exhibit similar antithrombin potency (Ki values), because of the differential compositional variations their anticoagulant effects in whole blood systems may differ due to their cellular and plasmatic protein interactions. Branded and generic versions of argatroban may exhibit differential anticoagulant actions in the whole blood and plasma based assays due to their differential interactions with blood cells, platelets and plasma proteins. Three generic versions of argatroban that are commercially available in Japan namely Argaron, Gartban and Slovastan and a powdered version of generic argatroban (Lundbeck) were compared with the branded argatroban. Native whole blood thrombelastographic (TEG) analysis was carried out at 0.1 ug/mL, the Activated Clotting Time (ACT) assay was carried out in a concentration range of 0–10 ug/mL, and such coagulation tests as the PT/INR, aPTT, Heptest, and calcium thrombin time were performed. Plasma retrieved from the supplemented whole blood was also assayed. Ratios of the clotting time test values from whole blood and plasma were calculated. Retrieved plasma samples were also assayed in the thrombin generation assays (TGA). All of the different versions of argatroban produced a concentration dependent anticoagulant effect in the native whole blood TEG and ACT. In the TEG, while argatroban and Slovastan showed a similar effect, Gartban, Argaron and a powdered generic showed weaker effects. Argatroban was also different in the ACT assay. At a concentration of 5 ug/ml the ACTs were, Arg 340+15.2 secs, S 297+10.5 secs, G 292.0+19.1 secs and A 285.2+21.7 secs. In the citrated whole blood systems, all agents produced a concentration dependent anticoagulant effect; however, the generic versions produced a stronger anticoagulant effect in comparison to branded argatroban (p<0.001). In the PT assay at 5 ug/mL, argatroban showed 32 ± 3 sec vs 40–50 sec for the generic products. Similarly in the aPTT, Heptest and thrombin time tests argatroban was weaker than the generic products. Differences among generic versions were also evident. Similar results were obtained in the retrieved plasma, however the ratio of whole blood over plasma varied from product to product. The IC50 of the generic and branded argatrobans in the TGA were also different. These results show that while in the thrombin inhibition assays generic and branded argatroban may show similar effects, these agents exhibit assay dependent differences in the whole blood and plasma based assays. Such differences may be more evident in the in vivo studirs where endothelial cells and other interactions may contribute to product individuality. Therefore, based on the in vitro antiprotease assays, generic argatrobans may not be considered equivalent and require a multi-parametric study. Currently available generic argatrobans may not be equivalent in the in vivo anticoagulant effects. Therefore, clinical validation of the clinical equivalence for these drugs is warranted. Disclosures: No relevant conflicts of interest to declare.

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Inhibition of Plasmin Generation in Plasma By Heparin, Low Molecular Weight Heparin and a Covalent Antithrombin-Heparin Complex (ATH)

Blood ◽

10.1182/blood.v124.21.4217.4217 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4217-4217

Author(s):

Gabriela Chang ◽

Helen M. Atkinson ◽

Leslie R. Berry ◽

Anthony K.C. Chan

Keyword(s):

Molecular Weight ◽

Plasminogen Activator ◽

Low Molecular Weight Heparin ◽

Conflicts Of Interest ◽

Area Under The Curve ◽

Low Molecular Weight ◽

Plasminogen Activator Inhibitor 1 ◽

Significant Difference

Abstract Introduction: Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are widely used anticoagulants for thrombosis treatment. However, these anticoagulants have limitations such as increased bleeding, variable dose response, required frequent monitoring, and, in the case of LMWH, inability to inhibit thrombin. This has led to the development of a covalent complex of antithrombin and heparin (ATH), which has been shown to overcome many of these shortcomings. ATH has faster rates of inhibition of many coagulation factors, is able to inhibit clot-bound thrombin, and is a more effective inhibitor of both venous and arterial thrombosis in animal models. Moreover, in a rabbit thrombosis model, ATH has been shown to decrease clot mass and fibrin accretion, while the contrary was observed for UFH. From these observations, it was suggested that ATH may enhance fibrin breakdown and thus led to investigations into the effects of UFH and ATH on fibrinolysis. In vitro studies have shown that UFH enhances antithrombin inhibition of plasmin. In addition, ATH displays a slightly greater inhibition of plasmin generation and activity. Such studies were conducted in purified systems, in the absence of other plasmin inhibitors naturally present in plasma. Therefore, the aim of the present study was to compare the effects of UFH, LMWH, and ATH on plasmin generation in plasma. Methods: At 37°C tissue plasminogen activator (tPA) and soluble fibrin fragments (fib) were added to normal adult pooled platelet poor plasma supplemented with 0.35, 0.7, 1.4, or 2.1 U anti-Xa/ml UFH, LMWH, or ATH, to initiate plasmin generation (8.93nM tPA and 300µg/ml fib). At various time points, subsamples were mixed with excess plasminogen activator inhibitor 1 (PAI-1) (55.12nM) to stop further plasmin generation. The plasmin concentration at each time point was determined using a plasmin-specific chromogenic substrate and a standard curve produced from purified plasmin. Results: Comparisons of mean area under the curve (AUC) for plasmin generation displayed a significant decrease in plasmin generation in the presence of all three anticoagulants at all doses tested (p<0.05). Comparing the anticoagulants at similar doses, plasmin generation was significantly decreased in the presence of ATH (15384.66±1930.23nM/min) compared to LMWH (23892.28±3090.54nM/min) at 0.7 U/ml (p<0.05). At a dose of 1.4 U/ml, there was significantly less plasmin generated, over time, in the presence of UFH (20089.49±3022.1623nM/min) and ATH (19273.86±1805.7323nM/min) when compared to LMWH (24743.18±1265.1023nM/min) (p<0.05). There was no significant difference in plasmin inhibition between UFH and ATH at any of the doses tested. Conclusion: The present study supports previous findings that UFH and ATH can facilitate antithrombin inhibition of plasmin. It is also observed that LMWH catalyzes the inhibition of plasmin by antithrombin but possibly to a lesser extent. These findings suggest that ATH has a similar inhibitory effect on plasmin generation and activity in plasma compared to UFH, despite its overall superior anticoagulant properties. Therefore, previous in vivo observations displaying decrease in clot mass with administration of ATH was due to its enhanced anticoagulant abilities and not fibrinolysis enhancement. These findings add to our understanding of ATH mechanisms of action and aid in its development for clinical use. Disclosures No relevant conflicts of interest to declare.

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Reversal of Factor Xa Inhibitors by Andexanet Alfa May Increase Thrombogenesis Compared to Pretreatment Values

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029619863493 ◽

2019 ◽

Vol 25 ◽

pp. 107602961986349 ◽

Cited By ~ 3

Author(s):

Fakiha Siddiqui ◽

Alfonso Tafur ◽

Lorenzo Storino Ramacciotti ◽

Walter Jeske ◽

Debra Hoppensteadt ◽

...

Keyword(s):

Thrombin Generation ◽

Area Under The Curve ◽

Factor Xa ◽

Coagulation Factor ◽

Inhibitory Effects ◽

Clotting Time ◽

Reversal Effect ◽

Normal Human ◽

Fxa Inhibitor ◽

Andexanet Alfa

Recombinant coagulation factor Xa (FXa), inactivated Zh-zo, also known as andexanet alfa (AA), is a modified version of human FXa that has been developed to neutralize FXa inhibitors. We studied the reversal effect of AA for these inhibitors in various anticoagulant and thrombin generation (TG) assays. Individual aliquots of normal human plasma containing 1 µg/mL of apixaban, betrixaban, edoxaban, and rivaroxaban, were supplemented with saline or AA at a concentration of 100 µg/mL. Clotting profiles include prothrombinase-induced clotting time, activated partial thromboplastin time, and prothrombin time. Factor Xa activity was measured using an amidolytic method. Thrombin generation was measured using a calibrated automated thrombogram. Differential neutralization of all 4 anticoagulants was noted in the activated clotting time and other clotting tests. The FXa activity reversal profile varied with an observed decrease in apixaban (22%), betrixaban (56%), edoxaban (28%), and rivaroxaban (49%). Andexanet alfa also led to an increased TG in comparison to saline. The peak thrombin was higher (40%), area under the curve (AUC) increased (15%), whereas the lag time (LT) decreased (17%). Andexanet alfa added at 100 µg/mL to various FXa supplemented systems resulted in reversal of the inhibitory effects, restoring the TG profile; AUC, LT, and peak thrombin levels were comparable to those of unsupplemented samples. Andexanet alfa is capable of reversing anti-Xa activity of different oral FXa inhibitors but overshoots thrombogenesis in both the saline and FXa inhibitor supplemented systems. The degree of neutralization of Xa inhibitor is specific to each agent.

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Reversion of the Experimental Hemodilutional Coagulopathy Induced by Crystalloids and Colloids Using Different Coagulation Factor Concentrates

Blood ◽

10.1182/blood.v118.21.4349.4349 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4349-4349

Author(s):

Carolina Caballo ◽

Ana M Galan ◽

Maribel Diaz-Ricart ◽

Irene Lopez-Vilchez ◽

Miguel Lozano ◽

...

Keyword(s):

Thrombin Generation ◽

Conflicts Of Interest ◽

Coagulation Factor ◽

Human Albumin ◽

Massive Bleeding ◽

Trauma Patients ◽

Beneficial Effects ◽

Ringer’S Lactate ◽

The Impact

Abstract Abstract 4349 BACKGROUND: Massive bleeding and subsequent coagulopathy are responsible for 35% of deaths in trauma patients. Hemodilution during resuscitation may worsen the coagulopathy and perpetuate bleeding. STUDY DESIGN AND METHODS: Blood samples from healthy donors were diluted (30–60%) using crystalloids (saline, Ringer’s lactate, Plasmalyte™) or colloids (6%hydroxyethylstarch (HES130/0.4), 5% human albumin, and gelatin). The impact of hemodilution on platelet adhesion, thrombin generation (TG), and clot viscoelastic properties by thromboelastometry (TEM) was analyzed. Effects of fibrinogen (Fbn), prothrombin complex concentrates (PCCs), rFVIIa, or cryoprecipates (cryo) on hemodilution were also assessed. RESULTS: Hemodilution caused a significant decrease in platelet interaction that was not improved by the addition of any of the plasma derivatives. A decrease in TG and important alterations of TEM were also observed. HES130/0.4 was the expander with the most deleterious action. TG was significantly enhanced by PCCs and their combination with Fbn whereas rFVIIa only slightly accelerated it. Fbn restored the alterations of TEM caused by hemodilution including those more deeply altered by HES 130/0.4. The combination of Fbn with PCC or rFVIIa did not have an additional effect in TEM. Cryo significantly improved the alterations caused by hemodilution on TG and TEM parameters. Effects of cryo on TG disappeared after ultracentrifugation, suggesting that contaminating microvesicular material could account for this effect. CONCLUSION: Hemostatic alterations caused by hemodilution are multifactorial and affect both blood cells and coagulation. In our in vitro approach, HES 130/0.4 seemed to exert a more deleterious effect on hemostasis. None of the concentrates improved platelet-mediated hemostasis, although they always showed variable beneficial effects on coagulation parameters. Our data indicate that PCC, rFVIIa and cryo enhance or accelerate thrombin generation. Fbn concentrates could be useful to preserve blood clotting abilities during fluid resuscitation of critically ill patients without exposing them to enhanced thrombin generation. Grants: PET(2008_0231), FIS(CP04-00112, PS09/00664), SAF2009-10365, RD06/0009 Disclosures: No relevant conflicts of interest to declare.

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Trace FVIII Augments Rfviia Induced Thrombin Generation and Improves Hemostasis in Hemophilia A Patients with Inhibitors: A clinical Cohort Study

Blood ◽

10.1182/blood.v120.21.40.40 ◽

2012 ◽

Vol 120 (21) ◽

pp. 40-40

Author(s):

Tami Livnat ◽

Uri Martinowitz ◽

Shirley Azar-Avivi ◽

Ariela Zivelin ◽

Gili Kenet

Keyword(s):

Thrombin Generation ◽

Hemophilia A ◽

Conflicts Of Interest ◽

Ex Vivo ◽

Therapy Response ◽

Response To Therapy ◽

Individual Therapy ◽

Severe Bleeding ◽

Inhibitor Level

Abstract Abstract 40 Treatment of Hemophilia A patients with inhibitors is challenging, as correlation between inhibitor level and hemostatic response to therapy may be limited. Thrombin generation (TG) assays may be used to monitor hemostasis and/or predict patients' response to various bypass agents. Since combination of excess FVIII and bypassing agents may potentiate improved TG in inhibitor plasma tested in-vitro, we aimed to define the therapeutic feasibility of co-administration of rFVIIa and FVIII in hemophilia A patients with inhibitors. Patients and Methods: Following consent, blood was sampled from 15 hemophilia patients (age: 0.5–46y) with inhibitor (0.5–668 BU). Platelet poor plasma (PPP) was prepared, spiked and incubated with excess FVIII. Ex-vivo kinetics of FVIII neutralization over time was evaluated by sequential measurements of residual FVIII activity. We then used recalcification induced-TG (performed in PPP supplemented with 4μM phospholipids), to measure the ex-vivo response to increasing concentrations of FVIII (0–200%) and rFVIIa (0–6.8μg/ml), alone or in combination. Based upon these ex-vivo studies, an individually tailored therapeutic regimen of concomitant bolus doses of rFVIIa and FVIII was applied to nine hemophilia patients with inhibitors. Results: FVIII ex- vivo measurements post incubation detected either rapid or slow neutralization- not correlating with inhibitor level. Flat baseline TG curves were recorded for all inhibitor patients, with variable responses to FVIII and/or rFVIIa. Combined spiking with FVIII and rFVIIa dramatically increased rFVIIa induced ETP (762.7 ±305.7 as compared to 339.3±179.9 nM/min with rFVIIa only) and peak height (48.7±23.6 vs 23.7±16.6) in all patients' plasma samples. Based upon individual ex vivo assays, concomitant bolus doses of rFVIIa (120–200 mcg/kg) and FVIII (50–100 U/Kg), were applied to 9 patients, for a total of 333 episodes during study period (February 2010-Septemeber 2012). Patients during immune tolerance received rFVIIa prophylaxis with combined rFVIIa/FVIII dosing applied 3 times weekly. For most mild- moderate joint bleeds hemostasis was defined as satisfactory following a single combined dose. Severe bleeding episodes or target joint bleeds responded to 2–8 (median:3) combined doses, applied every 12 hours. During study period the median number of spontaneous joint bleeds decreased from 4 to 1 per month. Neither thrombosis nor any other complications evolved. Conclusions: Prediction of individual therapy response may be achieved by pre-analytical studies, assessing FVIII neutralization kinetics as well as ex-vivo TG responses to combined bypass/FVIII therapy. Such studies enabled treatment of inhibitor patients according to individually tailored regimens. We confirmed for the first time that the in- vitro advantage of combining FVIII and rFVIIa, indeed accounts for improved hemostasis and may safely be applied to inhibitor patients. Disclosures: No relevant conflicts of interest to declare.

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FXIIa Differentially Regulates Thrombin Generation during Steady State and Vaso-Occlusive Crisis in Sickle Cell Mice

Blood ◽

10.1182/blood.v128.22.162.162 ◽

2016 ◽

Vol 128 (22) ◽

pp. 162-162 ◽

Cited By ~ 4

Author(s):

Erica M Sparkenbaugh ◽

Camille Faes ◽

Denis Noubouossie ◽

Daniel K. Kirchhofer ◽

András Gruber ◽

...

Keyword(s):

Steady State ◽

Mouse Model ◽

Vascular Permeability ◽

Sickle Cell ◽

Plasma Levels ◽

Thrombin Generation ◽

Conflicts Of Interest ◽

Control Group ◽

Liver And Kidney

Abstract Sickle cell disease (SCD) is associated with chronic activation of coagulation. Previously, we demonstrated that inhibition of tissue factor (TF) attenuates thrombin generation (measured by plasma levels of thrombin-antithrombin complexes [TAT]) in a mouse model of SCD during steady state. Furthermore, we showed that neither inhibition of FXIIa-dependent activation of FXI (using 14E11 antibody) nor FXI deficiency reduces thrombin generation (TG) in sickle mice. In contrast, genetic deficiency of FXII or kininogen (HK) reduced plasma TAT levels. These data suggest that during steady state, FXIIa contributes to TG in sickle mice via activation of the kallikrein/HK pathway, but not FXI. In the present study, we further investigated the mechanisms of HK-induced TG at steady state, and increased TG observed during vaso-occlusive crisis (VOC). All experiments were performed using 4-5 month old Townes SS (sickle) and AA (control) mice. Kallikrein cleaves HK into HK fragments (HKFs) and bradykinin (BK). First, we investigated whether a BK-mediated increase in vascular permeability contributes to TG by exposing perivascular TF. This hypothesis was disproved by data demonstrating no difference in vascular permeability (measured by the extravasation of Evans blue in the heart, lung, liver and kidney) between AA (n=8) and SS (n=10) mice. HKFs were shown to induce leukocyte TF expression in vitro via binding to CD11b/CD18 (Mac-1). Therefore, we investigated whether Mac-1 inhibition affects TG in SS mice. AA and SS mice were treated with an inhibitory anti Mac-1 (M1/70) or IgG control antibody on days 0, 3 and 6 (i.p. 1 mg/kg) and TG was analyzed 1 day after the last injection. In the control group, SS mice demonstrated higher plasma TAT levels compared to AA mice (8.1±1.6 vs 4.2±0.6 ng/mL, n=10-11, p<0.05), but inhibition of Mac-1 significantly reduced plasma TAT levels in SS mice (4.6±0.7 ng/mL, n=11, p<0.05). These data suggest that HK might contribute to TG during steady state via Mac-1-dependent induction of monocyte TF. The steady state of SCD is interspersed with acute periods of VOC. Clinical data demonstrate that compared to the steady state, plasma levels of cell free DNA (cfDNA), activation of the contact system, and TG are further enhanced during VOC. To determine the mechanism of increased TG during VOC, we used the previously characterized mouse model of TNFα -induced VOC. Townes AA and SS mice were injected with recombinant TNFα (2 µg/g body weight) or the same volume of PBS, and plasma was collected 5 hours later. TNFα not only dramatically increased plasma levels of cfDNA in SS mice (14.78 ± 1.64 vs 679 ± 300 ng/mL; p<0.01), but also further increased plasma TAT levels compared to those observed in PBS-treated SS mice (2.9 fold, p<0.001, n=8). Importantly, there was a significant positive correlation between cfDNA and TAT in SS mice (r2 =0.65, p<0.001). Since cfDNA can activate FXII, we determined whether FXIIa-dependent activation of FXI contributes to TG during VOC. AA and SS mice received 14E11 or IgG control (4 mg/kg) 30 minutes before TNFα (2 μg/g) or PBS injection, and plasma TAT was assessed 5 hours later. Strikingly, 14E11 attenuated the increased TAT level in TNFα-treated SS mice, to the level observed in SS mice injected with PBS and IgG (IgG/SS/PBS: 9 ng/mL ± 1.8 vs. IgG/SS/TNF: 18.9 ± 3.6, p<0.001; 14E11/SS/TNF: 9.86 ± 0.72, p<0.05 vs. IgG/SS/TNF). We also determined if TF activity is required for the increased TG observed during VOC. Interestingly, inhibition of TF with an inhibitory 1H1 antibody (25 or 75 mg/kg injected i.p. 1 or 18 hours prior to TNFα, respectively) had no effect on the increased TG observed in TNFα treated SS mice. In aggregate, our data suggest that during the steady state of SCD, FXII-dependent TG is not FXI-dependent, but instead is mediated by a pathway involving HK, Mac-1 integrin and leukocyte TF. Furthermore, we propose that during VOC the massive release of cfDNA results in FXIIa-dependent FXI activation and enhances TG independently of TF. This study provides mechanistic insight into the initiators of TG in SCD. Moreover, it implicates FXIIa as a potential therapeutic target to reduce the prothrombotic state in SCD, during both steady state and VOC. Disclosures No relevant conflicts of interest to declare.

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Decreased Thrombin Generation Potential Is Associated with Increased Thrombin Generation Markers in Sepsis Associated Coagulopathy

Blood ◽

10.1182/blood-2018-99-119675 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2505-2505

Author(s):

Fakiha Siddiqui ◽

Rajan Laddu ◽

Debra Hoppensteadt ◽

Matthew T. Rondina ◽

Yevgeniy Brailovsky ◽

...

Keyword(s):

Thrombin Generation ◽

Conflicts Of Interest ◽

Area Under The Curve ◽

Lag Time ◽

Coagulation System ◽

Clotting Factors ◽

Blood Samples ◽

Commercial Supplier ◽

Healthy Control ◽

Overland Park

Abstract Introduction: Sepsis associated coagulopathy (SAC) is commonly seen in patients which eventually leads to dysfunctional hemostasis and disseminated intravascular coagulation (DIC). Thrombin generation plays an important role in the overall pathophysiology of this process. Previous studies have reported an increase in thrombin generation markers such as thrombin antithrombin complex (TAT) and prothrombin fragment (F1.2). (Hoppensteadt et al. Clin Appl Thromb Hemost. 2014 Mar;20(2):129-35). Sepsis eventually results in consumption coagulopathy in which some of the clotting factors are consumed. The purpose of this study is to determine the thrombin generation potential of baseline blood samples obtained from sepsis associated coagulopathy patients and demonstrate their relevance to thrombin generation markers. Materials and Methods: Baseline citrated blood samples were prospectively collected from 49 patients with sepsis and suspected DIC. DIC scores were determined according to the ISTH scoring system (PTINR, fibrinogen, D-dimer and platelet count). Healthy control (n=50) represented citrated plasma obtained from a commercial supplier (George King Biomediacl, Overland Park, KS). Thrombin generation studies were carried out using a commercially available florigenic substrate methods with thrombin calibrator and PPP reagent (calibrated automated thrombogram; CAT). Such parameters as peak thrombin, lag time and area under the curve were compiled. TAT and F1.2 were measured using commercially available ELISA methods (Seimens, Indianpolis, IN). Functional antithrombin levels were measured using a chromogenic substrate method. All results were calculated in terms of mean ± SD. Applicable statistical methods were used to correlate the thrombin generation parameters with thrombin generation markers and antithrombin. Results: The peak thrombin levels were lower (82±40nM) in the SAC patients in comparison to higher levels observed in the normal plasma (133±10nM). The AUC was lower (561 ±280) in the SAC group in comparison to the normal (624±18). The SAC group showed much longer lag time (4.1±2.1) in comparison to the normal (2.1 ± 2.2). Wide variations in the results were observed in these parameters in the SAC group. The compsoite data is compiled in the table. the F1.2 levels in the DIC group were much higher (570±48 pmol) in comparison to the normal (210±25 pmol). The TAT levels also increased in the SAC group (27.9 ±5.1 ng/ml) in comparison to the normal (2.8 ±0.8 ng/ml). The functional antithrombin levels were decreased in the SAC group (64 ±11%). No correlation was observed between thrombin generation parameters and thrombin generation markers. Conclusion: These results validate the earlier observations that thrombin generation such as F1.2 and TAT are decreased in patients with SAC. However thrombin generation parameters are significantly elevated in this group in comparison to normals. This may be due to the consumption of prothrombin due to the activation of the coagulation system. The SAC group also showed wide variations in both the thrombin generation parameters and the F1.2 and TAT. These variations may be due to the differences in the endogenous pathophysiology state in the SAC patients. The decreased functional AT levels observed in the SAC group are due to the formation of the complex between generated thrombin and antithrombin. Simultaneous profiling of thrombin generation and thrombin generation markers may be helpful in the risk stratification of these patients. Table. Table. Disclosures No relevant conflicts of interest to declare.

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Monitoring Compound-Related Effects on Coagulability in Rats and Cynomolgus and Rhesus Monkeys by Thrombin Generation Kinetic Measurement

International Journal of Toxicology ◽

10.1177/1091581820907324 ◽

2020 ◽

Vol 39 (3) ◽

pp. 207-217

Author(s):

F. Poitout-Belissent ◽

D. Culang ◽

D. Poulin ◽

R. Samadfan ◽

S. Cotton ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

In Vivo Studies ◽

Coagulation Cascade ◽

Dependent Manner ◽

Kinetic Assay ◽

Thrombin Generation Assay ◽

Coefficients Of Variation

Thrombin generation assay (TGA) is a sensitive method for the assessment of the global clotting potential of plasma. This kinetic assay can detect both hypocoagulable and hypercoagulable conditions: delayed or reduced thrombin generation leading to a prolonged clotting time, or induced thrombin activity, shifting the coagulation cascade toward thrombosis. The purpose of this study is to qualify the TGA in nonhuman primates (NHP) and rats for its use during nonclinical in vivo and in vitro studies. Blood was drawn from nonanesthetized animals, and platelet-poor plasma was obtained after double centrifugation; coefficients of variation were <10% for all derived parameters of thrombin generation assessed with 5 pM of tissue factor. Thrombin generation was evaluated in vitro in rat and NHP plasmas with ascending doses of unfractionated heparin (UFH), recombinant tissue factor, and anticoagulant compounds. Thrombin generation was decreased with UFH and anticoagulant compounds, but was increased in the presence of tissue factor, in a dose-dependent manner. In a rat model of inflammation, animals were administered a low dose of lipopolysaccharides. Thrombin generation measurements were decreased 3 hours post-LPS administration with a nadir at 24 hours, while thrombin–antithrombin complexes reached a peak at 8 hours, supporting an earlier production of thrombin. In conclusion, these data demonstrated that TGA can be performed in vitro for screening of compounds expected to have effects on coagulation cascade, and thrombin generation can be measured at interim time points during nonclinical in vivo studies in rats and NHP.

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Comparative Studies on the Anticoagulant Profile of Branded Enoxaparin and a New Biosimilar Version

Clinical and Applied Thrombosis/Hemostasis ◽

10.1177/1076029620960820 ◽

2020 ◽

Vol 26 ◽

pp. 107602962096082

Author(s):

Dalia Qneibi ◽

Eduardo Ramacciotti ◽

Ariane Scarlatelli Macedo ◽

Roberto Augusto Caffaro ◽

Leandro Barile Agati ◽

...

Keyword(s):

Molecular Weight ◽

High Performance ◽

Thrombin Generation ◽

Area Under The Curve ◽

Factor Xa ◽

In Vivo Studies ◽

In Vitro Tests ◽

Thrombin Time

Low molecular weight heparins (LMWH) represent depolymerized heparin prepared by various methods that exhibit differential, biochemical and pharmacological profiles. Enoxaparin is prepared by benzylation followed by alkaline depolymerization of porcine heparin. Upon the expiration of its patent, several biosimilar versions of enoxaparin have become available. Heparinox (Sodic enoxaparine; Cristália Produtos Químicos Farmacêuticos LTDA, Sao Paulo, Brazil) is a new biosimilar form of enoxaparin. We assessed the molecular weight and the biochemical profile of Heparinox and compared its properties to the original branded enoxaparin (Lovenox; Sanofi, Paris, France). Clotting profiles compared included activated clotting time, activated partial thromboplastin time (aPTT), and thrombin time (TT). Anti-protease assays included anti-factor Xa and anti-factor IIa activities. Thrombin generation was measured using a calibrated automated thrombogram and thrombokinetic profile included peak thrombin, lag time and area under the curve. USP potency was determined using commercially available assay kits. Molecular weight profiling was determined using high performance liquid chromatography. We determined that Heparinox and Lovenox were comparable in their molecular weight profile. Th anticoagulant profile of the branded and biosimilar version were also similar in the clot based aPTT and TT. Similarly, the anti-Xa and anti-IIa activities were comparable in the products. No differences were noted in the thrombin generation inhibitory profile of the branded and biosimilar versions of enoxaparin. Our studies suggest that Heparinox is bioequivalent to the original branded enoxaparin based upon in vitro tests however will require further in vivo studies in animal models and humans to determine their clinical bioequivalence.

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Persistant Inhibition of Thrombin Generation After Intravenous Administration of Enoxaparin in Primates.

Blood ◽

10.1182/blood.v114.22.2095.2095 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2095-2095

Author(s):

Evangelos Litinas ◽

Angel Gray ◽

Nasir Sadeghi ◽

Josephine Cunanan ◽

Debra Hoppensteadt ◽

...

Keyword(s):

Thrombin Generation ◽

Time Course ◽

Conflicts Of Interest ◽

Clinical Effects ◽

Plasma Samples ◽

Thrombin Inhibition ◽

Biologic Effects ◽

Antithrombotic Effects ◽

Sustained Inhibition ◽

Inhibition Of Thrombin

Abstract Abstract 2095 Poster Board II-72 The biologic half life (T12) of low molecular weight heparin (LMWH) is usually measured in terms of the circulating anti-Xa levels. Enoxaparin represents an unique LMWH whose biologic T12 is relatively longer than most LMWHs. Moreover, it is known that the antithrombotic effects of this agent last longer in comparison to the measurable circulating anti-Xa levels. Therefore besides the anti-Xa activity, additional non-measurable biologic effects are contributory to the clinical effects of this agent. Plasma based thrombin generation assays have recently become available to assess the effects of LMWHs such as enoxaparin. In these assays blood plasma samples are activated using different activators and the generated thrombin inhibition is measured. To measure the time course of thrombin generation inhibitory activity after an IV bolus dose of 0.5 mg/kg of enoxaparin into groups of primates (n=6-8), a commercially available thrombin generation method was employed (Technoclone, Vienna, Austria/DiaPharma, West Chester,OH). Blood samples were drawn from each of the primates injected at varying time points for up to 28 hours. A thromboplastin/phospholipids based reagent was used to generate thrombin and the results were recorded in terms of nm of thrombin formed. The baseline values ranged from 500-900 nm (710±60 nm), although a complete inhibition of thrombin generation was noted at 1 hour (24±8 nm), a slow and gradual reduction in the thrombin generation inhibition was noted with a T12 of 9 hours. Even at 28 hours after the administration of enoxaparin, sustained inhibition of thrombin generation was noted (30-50%). Interestingly, the circulating anti-Xa and anti-IIa activity gradually diminished to an almost non-detectable level at 6 hours. These studies suggest that enoxaparin produces antithrombotic actions by multiple mechanisms. Furthermore thrombin generation methods in plasma samples may provide a more sensitive assay for the monitoring of the effect of LMWH. Disclosures: No relevant conflicts of interest to declare.

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